S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

Inoue, Akira; Omori, Akira; Ichinose, Sachiyo; Takahashi, Ken­ichi; Kinoshita, Yosihiro; Mita, Shiro

doi:10.1046/j.1432-1327.2001.02267.x

Cited by 7 publications

(17 citation statements)

References 62 publications

(82 reference statements)

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“…Hence S1 proteins may be involved in the formation/reorganization of VFs, which are needed for cell migration. S1 proteins C2 and D2 act as hnRNA binding proteins in the nucleus (Inoue et al, 2001), and as presented in this study, appear to be associated with VFs in the cytoplasm. It is interesting to speculate whether the S1 proteins participate in the localization of a particular set of mRNAs on VFs, thereby facilitating the site-oriented production of proteins required for formation or reorganization of VFs.…”

Section: Discussionsupporting

confidence: 68%

“…ARL cells were solubilized in a standard SDS sample buffer (SDS and 2-mercaptoethanol were at 2% and 1% respectively) and the lysates sonicated and heated at 95°C for 5 minutes. Immunoblots of 9.5% SDS-PAGE gels were probed with McAb 351 (1:1000 dilution) and a horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000 dilution, Cappel) (Inoue et al, 2001). Bands were visualized on X-ray film (Hyperfilm ECL, Amersham) with an ECL detection kit (RPN 2106, Amersham) and their intensities were measured using an image analyzer (BioRad, Multi-Analyst).…”

Section: Preparation Of Proteins and Immunoblottingmentioning

confidence: 99%

“…They are resolved by SDS-PAGE, each as doublets: A1 (an apparent molecular mass of 74.5 kDa), A2 (69.5 kDa); B1 (47.4 kDa), B2 (46.5 kDa); C1 (43.9 kDa), C2 (42.8 kDa); D1 (40.8 kDa) and D2 (39.4 kDa). S1 proteins B-D are multifunctional hnRNP proteins (Inoue et al, 2001;Inoue et al, 2003). Among them, S1 proteins D2 and C2 are identified as CArGbox binding factor-A (CBF-A) (Kamada and Miwa, 1992) and its splicing isoform (GenBank accession number AJ238854) respectively, acting as positive (Bemark et al, 1998;Mikheev et al, 2000) or negative (Kamada and Miwa, 1992) transcriptional regulators.…”

Section: Introductionmentioning

confidence: 99%

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Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

Inoue

Watanabe

Tominaga

et al. 2005

Journal of Cell Science

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S1 proteins C2 and D2 are multifunctional hnRNP proteins acting as transcriptional regulators in the nucleus. Immunofluorescence staining of various cells in culture revealed that S1 proteins also occur in the cytoplasm, often in association with vimentin intermediate filaments (VFs). Here, we verified the association of S1 proteins with vimentin using vimentin-deficient cells, crosslinking and immunoprecipitation, and further investigated the biological significance of this association. S1 proteins on VFs, referred to here as S1 fibers, were lost in highly confluent cells, where cell proliferation and cellular metabolic activity greatly decreased owing to cell density-dependent arrest. However, the disappearance of S1 fibers was not related to these reduced activities, but to inhibited cell migration. Although undetected in cells of non-migratory tissues as well as in confluent cultured cells, S1 fibers were found in all migratory cells examined, such as cultured cells in scratch/wound experiments, blood neutrophils and monocytes, and fibroblasts engaging in tissue healing. In addition, S1 fibers reappeared even in confluent cells when VFs were induced to reorganize with okadaic acid. We propose that S1 proteins occur in association with VFs in migratory cells. Possible participation of S1 proteins in the formation/reorganization of VFs is discussed.

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Section: Discussionsupporting

confidence: 68%

Section: Preparation Of Proteins and Immunoblottingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

Inoue

Watanabe

Tominaga

et al. 2005

Journal of Cell Science

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“…Membranes were probed with Ha-ras specific antibody (Santa Cruz, CA), CBF-A polyclonal [14] or monoclonal antibody [15] using standard procedures. Blotted antigens were detected using chemiluminescence reagent (NEN DuPont, MA).…”

Section: Western Blot Analysismentioning

confidence: 99%

“…All tumors were classified as papillary or tubular adenocarcinomas by histopathology. Total proteins extracted from tumors and the corresponding normal mammary tissues were analyzed by Western blot analysis using either the McAb351 anti-CBF-A monoclonal antibody or a polyclonal antibody against CBF-A [14,15].…”

Section: Deregulation Of Cbf-a In Mammary Tumorsmentioning

confidence: 99%

Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas

Mikheev

Inoue

Jing

et al. 2004

Breast Cancer Res Treat

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We previously identified a positive transcriptional element identical to human Ha-ras response element (HRE) within the promoter of the rat Ha-ras gene. We further identified CArG binding factor A (CBF-A), a member of heterogeneous nuclear ribonuclear protein (hnRNP) gene family, as a trans-acting factor that binds the HRE sequence with high affinity in rat mammary carcinoma cells. To determine if activation of CBF-A plays a role in tumor development in vivo , we investigated CBF-A expression and binding activity in rat mammary tumors induced by N-methyl-N-nitrosourea. We found that approximately 82% of tumors expressed CBF-A at levels that were 3-20 fold higher than detected in normal mammary gland. Moreover, elevated CBF-A protein levels were invariably associated with increased binding activity to the HRE. CBF-A mRNA levels in tumors were on average elevated only two fold as compared to normal mammary gland, indicating that increased CBF-A protein levels in tumors resulted from both translational and/or post-translational regulation. The level of CBF-A expression in mammary tumors was independent of Ha-ras mutational status. Together, these findings indicated that deregulation of CBF-A contributes to mammary carcinogenesis via a mechanism that is distinct from its hnRNP functions in binding and post-transcriptional regulation of RNA.

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Identification of a novel AU-rich-element-binding protein which is related to AUF1

Dean¹,

Sully²,

Wait³

et al. 2002

Biochemical Journal

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The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-alpha and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-alpha AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-alpha ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.

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S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

Cited by 7 publications

References 62 publications

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas

Identification of a novel AU-rich-element-binding protein which is related to AUF1

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